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Pharmacology & Therapeutics 186 (2018) 114–129

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Pharmacology & Therapeutics

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Associate editor: B. Rodriguez

Cardiac repair by epicardial EMT: Current targets and a potential role forthe primary cilium

Jessica N. Blom, Qingping Feng ⁎Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

Abbreviations: BBS8, Bardet-Biedl syndrome 8; bFGF, bextracellular matrix; EGF, epidermal growth factor; EMT,follistatin-like 1; GSK3β, glycogen synthase kinase 3β; HAport; IGF, insulin-like growth factor; Jbn, Jouberin; lncRNA3; MCP-1, monocyte chemoattractant protein 1; MI, myoPI3K, phosphatidylinositol 3-kinase; PDGF, platelet deriveceptor tyrosine kinases; RXRs, retinoidX receptors; SCF, stbox transcription factor 18; VEGF, vascular endothelial gr⁎ Corresponding author at: Department of Physiology a

E-mail address: [email protected] (Q. Feng).

https://doi.org/10.1016/j.pharmthera.2018.01.0020163-7258/© 2018 Elsevier Inc. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Available online 17 January 2018
Despite therapeutic advances that have prolonged life, myocardial infarction (MI) remains a leading cause ofdeathworldwide and imparts a significant economic burden. The advancement of treatments to improve cardiacrepair post-MI requires the discovery of new targeted treatment strategies. Recent studies have highlighted theimportance of the epicardial covering of the heart in both cardiac development and lower vertebrate cardiacregeneration. The epicardium serves as a source of cardiac cells including smooth muscle cells, endothelialcells and cardiac fibroblasts. Mammalian adult epicardial cells are typically quiescent. However, the fetal geneticprogram is reactivatedpost-MI, and epicardial epithelial-to-mesenchymal transition (EMT) occurs as an inherentmechanism to support neovascularization and cardiac healing. Unfortunately, endogenous EMT is not enough toencourage sufficient repair. Recent developments in our understanding of the mechanisms supporting the EMTprocess has led to a number of studies directed at augmenting epicardial EMT post-MI. With a focus on the roleof the primary cilium, this review outlines the newly demonstrated mechanisms supporting EMT, the role ofepicardial EMT in cardiac development, and promising advances in augmenting epicardial EMT as potentialtherapeutics to support cardiac repair post-MI.
© 2018 Elsevier Inc. All rights reserved.

Keywords:Cardiac repairMyocardial infarctionEpithelial-to-mesenchymal transitionEpicardiumCardiac progenitor cells

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152. Myocardial infarction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1153. Approaches to improve healing post-MI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154. EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155. Factors driving EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1166. EMT and cardiovascular development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1207. Cardiac repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1218. EMT for post-MI repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

asic fibroblast growth factor; BMP, bonemorphogenetic protein; BMSCs, bonemarrow-derived hematopoietic stem cells; ECM,epithelial-to-mesenchymal transition; EndMT, endothelial-to-mesenchymal transition; EPDCs, epicardium-derived cells; Fstl1,, hyaluronic acid; HGF, hepatocyte growth factor; Hh, hedgehog; HIF-1α, hypoxia-inducible factor 1α; IFT, intraflagellar trans-s, long non-coding RNAs; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; MEG3, maternally expressed genecardial infarction; miRNAs, microRNAs; MMPs, matrix metalloproteinases; ncRNA, non-coding RNA; PCP, planar cell polarity;d growth factor; Ptch, patched; RA, retinoic acid; RALDH, retinaldehyde dehydrogenase; RARs, retinoic acid receptors; RTKs, re-em cell factor; Shh, sonic hedgehog; Smo, smoothened; Tβ4, thymosin beta-4; TGF-β, transforming growth factor beta; Tbx18, T-owth factor; Wnt, wingless-type integration site; Wt1, Wilms' tumor 1.nd Pharmacology, Schulich School of Medicine and Dentistry, University ofWestern Ontario, London, Ontario N6A 5C1, Canada.

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115J.N. Blom, Q. Feng / Pharmacology & Therapeutics 186 (2018) 114–129

1. Introduction

Cardiovascular diseases are the second leading cause of death inCanada and the leading cause of death worldwide (Statistics Canada,2011; World Health Organization, 2015). Specifically, myocardialinfarction (MI), often caused by coronary artery disease, is the mostcommon form of sudden cardiac death (Hayashi, Shimizu, & Albert,2015). There has been significant improvement in therapeutic optionsfor patients to treat MI and the subsequent disease associated withpost-MI cardiac compensation. As a result, the in-hospital mortalityof patients with acute MI has decreased from 29% to 3% in the last50 years (de Vreede et al., 1991;Wang, Goodman, et al., 2015). Despitetherapeutic advancement, MI and post-MI morbidity pose a significantfinancial burden for the health care system of most nations. In Canada,MI is the third leading cause of hospitalization and a major driver of pre-scription drug use (Public Health Agency of Canada, 2009). The annualestimated direct and indirect cost of cardiovascular disease has recentlyamounted to over $21.2 billion in Canada alone (Tarride et al., 2009).The advent of percutaneous intervention aswell as the use of vasodilatorsand thrombolytics has markedly improved survival post-MI, however,approximately 26% of patients still progress to heart failure (Gerberet al., 2016). Furthermore, the number of patients forwhomconventionalrevascularization techniques fail is increasing. As such, the approach tothe treatment of heart failure is continuously evolving, and clinical trialscontinue to investigate emerging treatment options from stem cells togene therapy. Recently, the discovery that processes which occur duringthe development of the cardiovascular system are recapitulated in thedisease setting has directed research efforts towards modulating theseprocesses to enhance tissue repair. Epithelial-to-mesenchymal transition(EMT) is one such process that is up-regulated in the setting of MI whichsupports neovascularization and cardiac healing.

2. Myocardial infarction

The ventricular response to MI consists of a dynamic remodelingprocess which is often deleterious. Acutely, MI results in tissue damageand cardiomyocyte loss by both apoptosis and necrosis. Myocyte celldeath causes cytotoxic spilling and the recruitment of inflammatorycells occurs in the first three or four days post-infarction. In the sametime period, both resident and recruited fibroblasts and myofibroblastsproduce collagen (types I and III) to form a reparative fibrotic scar (vanden Borne et al., 2010). This initial scarring is paramount to the healingprocess as it prevents rupture of the ventricular wall. Myofibroblastsproduce inflammatory cytokines, and exhibit properties of bothfibroblasts and smooth muscle cells, thereby contributing to both scarformation and contraction of the granulation tissue. As the scar beginsto mature in the early phase of remodeling, more cells are graduallylost by apoptosis and necrosis, and characteristic mural thinning occurs.Over weeks to months, thinning and expansion cause ventriculardilation, which is initially beneficial, compensating for a reduced strokevolume. Ejection fraction, however is persistently depressed and enddiastolic pressure continues to rise. Consistent with the Frank-Starlingmechanism, reactive compensatory hypertrophy of remaining viablecardiomyocytes occurs. This hypertrophic response allows individualmyocytes to work harder to conserve ventricular function, but at acost of requiring additional blood supply to sustain cell metabolism.An essential component of the remodeling process is neoangiogenesiswithin the infarct region. Upregulation of pro-angiogenic agents likevascular endothelial growth factor (VEGF) and basic fibroblast growthfactor (bFGF/FGF-2) within the ischemic area promotes endothelialcell migration and pericyte communication (Ware & Simons, 1997).However, under normal conditions the growing capillary networkcannot keep pacewith the growing demands of hypertrophiedmyocar-dium; this evokes further myocyte necrosis. Alongwith wall thickeningand necrotic cell death, the ventricle muscle stiffens further; the initialreparative fibrotic response becomes exaggerated and the addition of

reactive fibrosis causes excessive collagen production, contributing toprogressive ventricular dysfunction (Talman & Ruskoaho, 2016).

3. Approaches to improve healing post-MI

Over the last 50 years, major efforts in cardiac research have focusedon minimizing infarct expansion, fibroblast collagen deposition, andcardiac hypertrophy following MI. Methods such as inhibiting inflam-mation, reprogramming cardiacfibroblasts, activating cell cycle reentry,transferring skeletal myoblasts to the infarct region, and transplantingprogenitor cells have been tested (Christia & Frangogiannis, 2013;Ptaszek, Mansour, Ruskin, & Chien, 2012). These methods have beenmet with conflicting results in in vivo models and in the clinic. Earlystudies in murine models suggested that bone marrow-derived hema-topoietic stem cells (BMSCs) introduced into themyocardium followingMI could differentiate into cardiomyocytes and aid in repair; however,these cells did not differentiate or incorporate into the myocardium insubsequent studies (Murry et al., 2004). A number of clinical trialsusing autologous stem or progenitor type cells were initiated in theearly 2000’s, however meta-analyses of these trials show modestimprovements in cardiac function or no significant benefit onventricular remodeling (Delewi et al., 2014; Jeong et al., 2013; Liu,Duan, et al., 2014; Zhang et al., 2009). Many traditional therapies werealso directed at increasing infarct zone microvascularity and promotingangiogenesis. It is thought that if blood flow can be restored to theinfarcted or “hibernating” myocardium, fewer cardiomyocytes will belost. For example, augmented infarct zone angiogenesis has beendemonstrated in animal models using VEGF and bFGF therapy (Banaiet al., 1994; Yanagisawa-Miwa et al., 1992), however this was notassociatedwith benefit in human trials (Mitsos et al., 2012). To augmentneovascularization, research has turned to exploring processes whichsupport coronary artery formation during development, namely EMT.

4. EMT

EMT is a process by which epithelial cells undergo transcriptionalreprogramming to transdifferentiate into motile mesenchymal cells(Lamouille, Xu, & Derynck, 2014). Epithelial cells are characterized bydefined polarity which is maintained through intercellular junctionsincluding tight junctions, adherens junctions, desmosomes, and gapjunctions. EMT involves cell junction disassembly, loss of epithelial cellpolarity, and cytoskeletal reorganization via suppression of epithelialgenes, and activation of a mesenchymal gene signature. Mediators ofEMT are upregulated locally, and their expression and receptor bindinginitiates an intracellular molecular cascade which supports the cellphenotype switch. For example, the upregulated local expression oftransforming growth factor beta (TGF-β) and bFGF activated kinasesand small GTPases facilitates degradation of the basement membraneand promotes actin rearrangement and cell movement (von Gise & Pu,2012). TGF-β stimulates the expression of Snail and Slug, transcriptionfactors which directly repress transcription of epithelial genes.Epithelial E-cadherin is a transmembrane protein that forms epithelialcellular junctions; specifically, E-cadherin is critical to the adhesionbelt of adherens junctions (Hartsock &Nelson, 2008). Upon EMT activa-tion, E-cadherin is both cleaved at the cell membrane and endocytosed,and its production is suppressed in favor of N-cadherin, a mesenchymalprotein (David & Rajasekaran, 2012; Lamouille et al., 2014). This“cadherin switch” is a hallmark of EMT, and supports the gain of affinityfor mesenchymal cells rather than endothelial cells.

Cadherin complexes are connected to the actin cytoskeleton bymem-bers of the catenin family. P120-catenin, α-catenin, and β-catenin stabi-lize E-cadherin at the plasma membrane (Hartsock & Nelson, 2008).However, the dual role of β-catenin implicates it in both themaintenanceof an epithelial phenotype and induction of mesenchymal transition. Se-questration of β-catenin at the adherens junction supports maintenanceof junction integrity, however the degradation of E-cadherin liberates

116 J.N. Blom, Q. Feng / Pharmacology & Therapeutics 186 (2018) 114–129

β-catenin, allowing its cytoplasmic localization. Wingless-type integra-tion site (Wnt) pathway activation (discussed later) causes β-cateninnuclear translocation and transcriptional activation of Wnt/β-catenintarget genes to support EMT (Valenta, Hausmann, & Basler, 2012).

In addition to the cadherin switch, cellular motile capacity isenhanced by actin reorganization, which enables extension of thecytoskeleton. Lamellipodia and filopodia extend from the plasmamem-brane, and proteolytically degrade the extracellular matrix (Ridley,2011). Meanwhile, actin is polymerized in the direction of migration(Olson & Sahai, 2009). Further facilitating migration, the intermediatefilament composition switches from cytokeratin-rich to vimentin-rich(Mendez, Kojima, & Goldman, 2010). The cell assumes a mesenchymalphenotype and migrates away from the basement membrane. AfterEMT, cells exhibit a mesenchymal gene signature, and are able tore-differentiate into other cell types.

5. Factors driving EMT

There are numerous molecules implicated in a wide range ofoverlapping signaling pathways that support EMT. Among others,

EGFIGFHGF

RTKsTGFRTGF-βBMP

BMPR

SMADP

TFs

AKT/PKB

MEK1/2

HYPOXIA

HIF1-α

PI3-K

PP

Tβ4

ERK1/2

RAR/R

PDGFFGF

Fig. 1. Signaling pathways mediating EMT. TGF-β and BMP both phosphorylate SMAD proteinsMAPK pathways which are known for being regulated by ligands of RTKs, including PDGF, FGF,which is inhibited by canonical Wnt signaling. Shh promotes the nuclear translocation of EMmembrane bound ligand DLL or Jagged from a neighboring cell to cause nuclear transport oreceptors (RAR and RXR), which themselves act as transcription factors. In addition to ligupregulated in EMT to support mesenchymal like cytoskeletal changes. The GTPase RhoA issupports the entire process by upregulating transcription of HIF-1α, which in turn binds hypo

these include TGF-β, bFGF, hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), Notch,Wnt, retinoic acid (RA), hypoxia inducible factor (HIF-1α), and Sonichedgehog (Shh). The mechanisms of by which these molecules supportEMT is outside the scope of this review, but has previously beendiscussed in the following eloquent reviews (Lamouille et al., 2014;von Gise & Pu, 2012). Another important factor modulating EMT isthymosin beta-4 (Tβ4), an actin-monomer-binding protein known toregulate cell motility (Mannherz & Hannappel, 2009). Most of thepathways through which these molecules exert their functionsconverge on the transcriptional control of epithelial-specific andmesenchymal-specific proteins, including E-cadherin. Regardless ofthe predominant EMT pathway upregulated to provoke the initiationof signaling, common downstream effectors of these pathways are tran-scriptional repressors like Snail, Slug, ZEB1/2 and Twist, which block theE-cadherin promoter region (at E-box elements), resulting in a mesen-chymal switch (Germani, Foglio, Capogrossi, Russo, & Limana, 2015;Lamouille et al., 2014). Signaling pathways supporting EMT aresummarized in Fig. 1. The focus of this review is on non-coding RNAand the primary cilium.

Ptch1Shh

FzWnt

SlugSnail1

β-CateninTwist

ZEB1/2

Notch

NCID

EMT

Cytoskeletal change

Dvl

Smo

Gli1/2

GSK3β

DLL/Jag

RhoA

XR

β-Catenin

RA

RA

, leading to a SMAD phosphorylation cascade. TGF-β also activates the PI3K/AKT and ERK/EGF, IGF, and HGF. RTKs inhibit the activity of the destruction complex containing GSK3β,T transcription factors by binding its receptor Ptch. The Notch receptor is activated by itsf its intracellular domain. Furthermore, RA diffuses through the cell to bind its nuclearand mediated EMT, other mediators, such as the actin sequestering protein Tβ4 arealso activated by Tβ4 and TGF-β, and regulates changes in cell shape. Finally, hypoxia

xia response elements of other genes to cause EMT.


5.1. Non-coding RNA

EMT is a highly regulated event, which proceeds through awide spectrum of intermediates. Genome wide analysis studieshave determined that although the majority of the human (andmouse) genome is transcribed (80%), the bulk (62–75%) of thesetranscripts do not code for proteins (Djebali et al., 2012; EncodeProject Consortium, 2012). Questioning the doctrine of the “centraldogma” theory, non-coding RNA (ncRNA) genes produce RNAmolecules that are functional in and of themselves and are not merelyintermediary molecules that encode proteins. MicroRNAs (miRNAs;18–24 nucleotides) and long non-coding RNAs (lncRNAs; N200nucleotides) are two subsets of ncRNA which modulate EMT bytranscriptional repression, epigenetic modification, and translationalregulation.

miRNAs repress translation by binding the 3′-untranslated regionof target mRNAs. Ibrahim et al. (2017) recently demonstrated theimportance of miRNAs in EMT by altering the activity of Dicer, theRNase enzyme responsible for cleaving pre-miRNA into miRNA. Forcedexpression of Dicer prevented hypoxia-induced increase in HIF-1αexpression, and reduced cell migration and EMT in hepatocellularcarcinoma cells. Furthermore, several miRNAs have been reported tomodulate EMT transcription factors and protein components of thecell architecture. One of themost well studied miRNA families involvedin EMT is the miR-200 family (miR-200a, miR-200b, miR-200c,miR-141, and miR-429), reviewed by Korpal and Kang (2008). Severalstudies have demonstrated that members of the miR-200 family targettranscriptional repressors of E-cadherin and moderate EMT drivenby Wnt/β-catenin. (Cong et al., 2013; Korpal, Lee, Hu, & Kang, 2008;Su et al., 2012). Intriguingly, PDGF mediated EMT is augmented inthe presence of anti-miR-200 and diminished upon anti-miR-221treatment (Kong et al., 2009; Su, He, Tian, Hu, & Zhang, 2013). Thecritical EMT transcription factor Snail is also mediated by miRNAs,as demonstrated via 3'UTR binding and translational inhibition bythe miR-30 family (Kumarswamy et al., 2012). miRNAs also targetcomponents of the cell architecture, thereby affecting epithelialintegrity. For example, miR-9 directly targets E-cadherin and supportsrelease of β-catenin and mesenchymal transformation in mammaryepithelial cells (Ma et al., 2010). The molecular mechanisms bywhich miRNAs act as critical regulators in determining the fate oftransitioning cells are described in recent reviews (Abba, Patil,Leupold, & Allgayer, 2016; Lamouille, Subramanyam, Blelloch, &Derynck, 2013).

More recently, studies have begun to illuminate the role of lncRNAsin EMT. The lncRNAs comprise the largest portion of the non-codingtranscriptome, with 96,308 genes and 172,216 transcripts discoveredin the human to date (NONCODE database, http://www.noncode.org/analysis.php). A number of lncRNAs have been described to regulateEMT, with some possessing supportive functions, and others repressivefunctions. For example, the lncRNA metastasis-associated lung adeno-carcinoma transcript 1 (MALAT1) has been described to control EMT,apoptosis, cell cycle progression, and angiogenesis (Michalik et al.,2014; Xiang et al., 2016; Xiang, Zhang, Tang, & Li, 2017). MALAT1localizes to nuclear bodies (nuclear speckles) within the nucleus,where it is suggested to regulate alternative splicing of several pre-mRNAs (Tripathi et al., 2010). Indeed,MALAT1was originally identifiedas a marker for non-small cell lung cancers with metastatic propensity(Ji et al., 2003). MALAT1 transcripts have also been found to beupregulated in numerous cancer types and it is thought to contributeto the maintenance of EMT for cell migration and invasion (Gutschner,Hammerle, & Diederichs, 2013; Zhou et al., 2015). Inhibition ofMALAT1 in both in vivo and in vitro models of squamous cell carcinomareduced cell migration and invasion, expression of EMT markers Slugand β-catenin, and the appearance of mesenchymal markers N-cadherin and Vimentin (Zhou et al., 2015). Furthermore, Yang, Yao, Li,Li, and Wang (2016) demonstrated a direct link between MALAT1 and

TGF-β-induced EMT in human retinal pigment epithelial cells in whichknockdown of MALAT1 using siRNA suppressed TGF-β-mediated mor-phological changes, α-SMA expression, as well as the down-regulationof E-cadherin. The role of MALAT1 in epicardial EMT has yet to beelucidated.

Contrastingly, the lncRNA maternally expressed gene 3 (MEG3),recently identified through genome-wide mapping, suppresses bothTGF-β and SMAD as well as TGF-β receptor expression by binding GArich sequences (Mondal et al., 2015). Furthermore, MEG3 was shownto specifically repress EMT in breast cancer cells in a transwell invasionassay; MEG3 was demonstrated to sponge miRNA-421 to maintainE-cadherin expression (Zhang et al., 2017). As a result, decreasedexpression ofMEG3 is associated with poor prognosis in cancer patients(Sun et al., 2014). Interestingly, two groups have recently demonstrateda role forMEG3 in cardiac tissue. In experiments by Piccoli et al. (2017),GapmeR-mediated silencing of MEG3 in cardiac fibroblasts reducedMMP-2 transcription. Inhibition of MEG3 in vivo prevented transverseaortic constriction induced cardiac fibrosis and cardiomyocyte hyper-trophy. Similarly, Gong et al. (2018) recently identified a deleteriousresponse to MEG3 expression in H9c2 cells, demonstrating that MEG3shRNA mediated knockdown alleviates hypoxia-induced cell injury. Acareful investigation of mechanisms of MEG3 action in different celltypes and for different cardiac pathologies are warranted in futureresearch. Beyond those mentioned here, a number of lncRNAs areinvolved in regulating EMT, as is eloquently reviewed by Heery, Finn,Cuffe, and Gray (2017).

5.2. The primary cilium

Outside of signalingmediators, cellular structural changes may alterreceptor responses and thus downstream effects of EMT signals.Receptors for cytokines that govern multiple signaling pathwaysintegral to the initiation of EMT are found on small microtubule basedsensory organelles called primary cilia (Koefoed, Veland, Pedersen,Larsen, & Christensen, 2014). Primary cilia are non-motile hair-likeprotrusions that emanate from the cell surface in an antenna-likefashion. Primary cilia are located on almost all polarized, and mostnon-polarized cell types (Davenport & Yoder, 2005). These organellespossess unique antennal like properties which allow them to detectmechanical and sensory cues and transmit these signals from the cellsurrounding to its interior.

The ciliary axoneme is elongated, and continuously turned over viathe process of intraflagellar transport (IFT). IFT is a bi-directional trans-port systemwhich allows the trafficking of large protein complexes (IFTparticles) along polarized microtubules in the axoneme (Kozminski,Johnson, Forscher, & Rosenbaum, 1993). The IFT complexes serve asrafts for ciliary cargo such as tubulin, membrane proteins, ion channels,and protein kinases (Lechtreck, 2015). Dysregulation of IFT polypep-tides leads to a plethora of human diseases, demonstrating theimportance of these molecules for proper ciliary function. These“ciliopathies” are often pleiotropic disorders and include defects innumerous organ systems including the liver, kidney, airway, brain,pancreas, retina, adipose, and heart (Davenport & Yoder, 2005). Forexample, intraflagellar transport protein 88 (IFT88) (Tg737; Polarisprotein) is a complex B protein required for primary cilia formation.This requirement was discovered in Chlamydomonas, and subsequentlydetermined to contribute to the failure of ciliary function in polycystickidney disease (Moyer et al., 1994; Pazour et al., 2000). Tg737 function-ality was also shown to be required for various ciliary related processesincluding proper ependymal cell function in brain ventricles and nodecilia functioning in left–right patterning for embryonic development(Taulman, Haycraft, Balkovetz, & Yoder, 2001). Tg737 knock-out micedie at embryonic day 11.5 due to a variety of developmental abnormal-ities, including cardiac insufficiency with pericardial sac ballooning(Murcia et al., 2000).

http://www.noncode.org/analysis.php

http://www.noncode.org/analysis.php


5.2.1. Wnt at the primary ciliumIFT allows the transport of signaling molecules and receptors,

turning the cilium into a repository that can harbor proteins andcoordinate signaling pathways. Various lines of evidence indicate thatthe primary cilium plays an inhibitory role in Wnt signaling. Knockoutmice for proteins required for primary cilia assembly exhibit hyperac-tive β-catenin-dependent signaling as compared to wild-type controls(Corbit et al., 2008). It is thought that the β-catenin-positive regulatorJouberin (Jbn) is spatially sequestered in the cilium, constrainingmovement of β-catenin to the nucleus. To this effect, Lancaster,Schroth, and Gleeson (2011) demonstrated ciliary targeting motifs onthe Jbn protein, and increased nuclear co-localization of Jbn and β-ca-tenin upon Kif3a siRNA mediated cilia retraction (Fig. 2). β-catenin isa known transcriptional activator of several EMT related genes,

Fig. 2. Signaling associated with the primary cilium. Wnt receptors are associated with the bascytoplasm and its nuclear transport is uninhibited, causing hyperactive signaling. Primary ciliusignaling. PDGFα receptors are also associated on the ciliumwhile PDGFβ receptors are only on teffect on PDGFβ signaling.

including Snail1 and Zeb1 (Yang et al., 2015). Unlike the canonical path-way, however, the primary cilium is thought to be required for planarcell polarity (PCP, non-canonical Wnt signaling), and core PCP compo-nents are depleted in IFT mutants (Cao, Park, & Sun, 2010). Similarly,Cre-mediated conditional mutation of Ift88 in the inner ear, results indisorganization of the planar-polarized orientation of stereocilia(Jones et al., 2008). Furthermore, several core PCP proteins, such asVangl2, have been shown to localize to the primary cilium axoneme(Borovina, Superina, Voskas, & Ciruna, 2010). May-Simera et al.(2010) demonstrated the requirement for the interaction betweenVangl2 with the ciliary/ basal body protein Bardet-Biedl syndrome8 (BBS8) for the establishment of left–right asymmetry. In this study,a direct interaction between Vangl2 and Ift20 was demonstrated byco-immunoprecipitation, and deletion of Ift20 interrupted PCP-

e of the primary cilium. In the absence of the cilium, Jouberin (Jbn) binds β-catenin in them also harbors sonic hedgehog (Shh) signaling factors, and its degradation decreases Shhheplasmamembrane. Loss of the ciliumattenuates PDGFαmediated responses, but has no


dependent asymmetric protein accumulation. On the other hand, usingFrizzled-2 knockdown, it has been demonstrated that mediators of non-canonical Wnt/PCP signaling are required for primary cilia formation(Oishi, Kawakami, Raya, Callol-Massot, & Izpisua Belmonte, 2006).Regardless of whether the primary cilium is required for noncanonicalWnt signaling, or vice versa, the two appear to be closely connected,perhaps via various molecules. In either case, mutations in ciliaryproteins attenuate Wnt/PCP signaling, resulting in loss of cell polarity –a feature of EMT (Lamouille et al., 2014).

5.2.2. Shh at the primary ciliumIn addition toWntmediators, Ptch, Smo, andGli transcription factors

are all present within the primary cilium. This localization of Ptch andSmo within the primary cilium is mutually exclusive (Haycraft et al.,2005; Rohatgi, Milenkovic, & Scott, 2007). In a basal state, Ptch islocalized to the primary cilium. In the absence of Shh, Ptch tonicallyinhibits Smo from its entrance into the cilium, thereby repressing Hhmediated transcription. In this instance, Smo cannot act on the Glifactors, and Gli2 is degraded while Gli3 is processed to its repressiveform. Alternatively, in the presence of Shh, Ptch is internalized, allowingSmo to enter the primary cilium (Fig. 2). Within the cilium, Smo sendsGli3 to the proteasome for degradation, and activates the Gli2 transcrip-tion factor, which enters the nucleus to promote transcription. Theinvolvement of the primary cilium in this process is demonstrated bythe requirement of IFT for both Gli2 activator and Gli3 repressor pro-cessing. Loss of IFT results in reducedGli2 activators andGli3 repressors,causing aberrantly active low level Hh signaling (Caspary, Larkins, &Anderson, 2007; Haycraft et al., 2005). During development, at whichtime Hh levels are high, disrupting IFT proteins results in neural tubeand brain defects (Huangfu et al., 2003). However, in postnatal tissues,the primary cilium inhibits Hh signaling. A loss of primary cilia mayactivate Hh signaling, which regulates plasticity of the pancreaticepithelium and may contribute to tumorigenesis (Cervantes, Lau,Cano, Borromeo-Austin, & Hebrok, 2010; Hassounah, Bunch, &McDermott, 2012).

5.2.3. PDGF at the primary ciliumThe primary cilium is essential for PDGF signaling in growth arrested

cells through its receptor PDGFRαα, the homodimer of PDGFRα(Schneider et al., 2005). PDGFRαα is localized to the primary cilium,and in Tg737orpk (or Ift88) knockout mutants, embryonic fibroblast cellcycle entrance was blocked. Reduced PDGFRα expression in thesecells led to decreased MEK1/2-ERK1/2 activation, delineating a role forthe primary cilium and PDGF signaling in growth control. The samegroup also used micropipette administration to demonstrate theimportance of this signaling pathway for directional wound healing,indicating that the primary cilium is required for chemotaxis towardsPDGF-AA (Schneider et al., 2010). Defects in PDGF signaling areimplicated in a range of diseases including epithelial cancers and vascu-lar disorders (Andrae, Gallini, & Betsholtz, 2008). For example, aberrantPDGF causes EMT and supports cancer metastasis, as demonstrated byreduced experimental metastasis with overexpression of a dominant-negative PDGFR (Jechlinger et al., 2006). PDGF signaling also plays arole in EMT of mesenchymal cells during cardiac development and thegrowth of the coronary vasculature (van den Akker et al., 2005).However, using immunohistochemistry, these authors demonstratedthe presence of PDGF-B and PDGFRββ, rather than PDGFRαα in EPDCsundergoing EMT for coronary artery development. Although Schneideret al. (2005) demonstrated that PDGF-AA signaling through PDGFRααis associated with the cilium, signaling through PDGFRββwas unaffect-ed by loss of the cilium (Fig. 2). Interestingly, activation of PDGFRββ hasbeen shown to promote deciliation, a phenotype associated withciliopathies. In experiments with NIH3T3 serum deprived cells, potentdeciliation was observed under immunofluorescence microscopywith the addition of PDGF-DD (a PDGFββ agonist), but not PDGF-AA(Nielsen et al., 2015). In these experiments, serum-induced deciliation

was markedly impaired with depletion of PDGFRβ using siRNA.Furthermore, isolated deletion of PDGFRβ (PDGFRβ−/−) results in animpairment of coronary vascular smoothmuscle generation in develop-ment, while knockout of PDGFRα alone inhibits the generation ofcardiac fibroblasts, differentiating the two PDGF receptors (Mellgrenet al., 2008; Smith, Baek, Sung, & Tallquist, 2011). Thus, the type ofPDGF may be an important consideration when determining the roleof the cilium in PDGF signaling. Specifically, it has been suggested thatsignaling through PDGFRαα requires the presence of the cilium, whilePDGFRββ leads to activation of the downstream MEK/ERK mediatorsindependent of the cilium. In this regard, addition of PDGF-BB toTg737orpk mouse embryonic fibroblasts caused ERK1/2 and AKTphosphorylation while PDGF-AA had no effect (Clement et al., 2013).Notably, PDGF-AA/αα signaling can be restored in ciliary transportmutants by inhibiting mTORC1 with rapamycin, suggesting that whilethe cilium is not absolutely required for PDGFαα signaling, it doesplay a key role in augmenting efficiency of PDGFααmediated responses(Umberger & Caspary, 2015).

5.2.4. Other signaling associated with the primary ciliumIn a recent review, Christensen, Morthorst, Mogensen, and Pedersen

(2017) eloquently described the role of the primary cilium in coordinat-ing both RTK and TGF-β signaling. In addition to their role in regulatingPDGF, primary cilia have been demonstrated to mediate the activity ofother RTKs, which autophosphorylate to activate MAPK and PI3K-AKTsignaling. The epidermal growth factor receptor (EGFR) is also localizedon the primary cilium (Ma, Li, et al., 2005), and loss of the primarycilium in Kif3a mutant mice results in apical mislocalization of theEGFR on the tubular epithelial cells (Lin et al., 2003). Although the roleof the primary cilium in signaling has not been elucidated, Seeley andNachury (2009) has suggested that loss of the IFTmotor complex causesEGFR accumulation inside the cell and resulting persistent MAPKactivity. In other cases, signaling through someRTKs appears to facilitatechanges in ciliary structure. For example, IGF-1 binds to IGF-1R on thecilia to support cilia resorption in retinal pigment epithelial cells (Yehet al., 2013). Interestingly, in this study, IGF-1R was found to be locatedboth on the cilium and on the plasma membrane. IGF-1 receptorneutralizing antibodies blocked ciliary resorption, and addition ofIGF-1 alone stimulated resorption and cell cycle reentry. The additionof IGF-1 to Ift88mutant cells which lack functional cilia did not supportsimilar mitogenic responses, suggesting that the cilium is required tomediate IGF-1 signaling. Conflictingly, the combination of both IGF-1,EGF or FGF and PDGF-AAwas required tomediate ciliary destabilizationin mouse embryonic fibroblasts (Jacoby et al., 2009). In this study,addition of IGF-1, EGF or FGF alone did not alter cellular architecture,however when one of these factors was added in combination withPDGF-A the cilium was disassembled, indicating a requirement forPDGF-A and permissive roles for the other RTKs in cilia disassembly.Notch receptors are also located on the primary cilium, however theinvolvement of the cilium in the regulation of the Notch pathway is asubject of debate. While some groups demonstrate a requirement forthe primary cilium in Notch signaling for early development (Ezrattyet al., 2011), others show over activation of the Notch pathway upondisruption of ciliary localization (Leitch, Lodh, Prieto-Echague, Badano,& Zaghloul, 2014).

Like RTKs and Notch, TGF-β receptors are located on the primarycilium, and primary cilia appear to be required for TGF-β dependentSMAD signaling (Clement et al., 2013). In this study, immunofluores-cence microscopy was used to demonstrate the localization of TGF-βreceptors to the tip of the cilium in fibroblasts, and the activated TGF-β receptors migrated to the ciliary base to phosphorylate SMAD2/3and activate ERK1/2 (Clement et al., 2013). These authors also demon-strated the requirement for the cilium in this process as TGF-βmediatedSMAD phosphorylation was significantly attenuated in fibroblasts fromTg737orpkmutant cells. In another study however, primary ciliawere notrequired for responses to TGF-β/BMP type ligands. In fact, non-ciliated


endothelial cells from Tg737orpk mutant embryos exhibited a fibroblastphenotype after exposure to fluid flow and enhanced phospho-Smad2and Snail1 expression as compared to ciliated cells (Egorova et al.,2011). Upon exposure to increased shear stress (2.5 Pa), ciliated endo-thelial cells became non-ciliated and EMT marker expression reflectedsimilar propensity for change as Tg737orpk mutant cells. In fact, rescueof the cilium in Tg737orpk mutant cells by Ift88 transfection inhibitedEMT. Similarly, Sanchez-Duffhues et al. (2015) demonstrated that endo-thelial cells lacking primary cilia are prone to undergo morphogenicchange in response to BMP; interestingly, BMP rather than TGF-β wasrequired for SMAD phosphorylation in cilium-defective aortic endothe-lial cells, and this supported β-catenin and Slug expression. Importantly,Rozycki et al. (2014) demonstrated that the resorption of the primarycilium is involved in regulating TGF-β mediated myofibroblasttransition. In this study, wounding to de-couple epithelial intercellularcontacts provoked initial ciliumgrowth, and subsequent TGF-β stimula-tion evoked cilium loss and EMT like transition. Provoking cilium losswith Kif3a siRNA prior to EMT made the cells insensitive to TGF-β,however if the Kif3a siRNA was applied with optimal timing for peakdeciliation after TGF-β stimulation, the ensuing cilium loss acceleratedthe EMT process. Thus, it is likely that ciliary signaling is both ligand,cell type, and condition specific. It is possible that mediators ofEMT bind receptors on the primary cilium to induce cilia resorptionand downstream signaling that supports EMT, and this process isfurther potentiated by the addition of other ligands post resorptionlike BMP.

6. EMT and cardiovascular development

EMT plays a critical role in early development. Beyond its role in theinvasion of the uterine lining and forming the three germ layers, EMT isalso responsible for cardiac formation. Originating in the proepicardialorgan, cells that express the transcription factors Wilms' tumor 1(Wt1) and T-box transcription factor 18 (Tbx18) in developmentmigrate to the surface of the myocardium to become the epicardiallayer at around E9.0–10.5 in mice (Cai et al., 2008; Vicente-Steijnet al., 2015). Many of these cells then undergo EMT to delaminatefrom the epicardium and form epicardium-derived cells (EDPCs)which migrate into the myocardium as the precursors of the cardiacstructures (about E11.5–12.5) (Vicente-Steijn et al., 2015). Specifically,EPDCs contribute to the subendocardial and atrioventricular cushion,cardiac interstitial fibroblasts, and the coronary vasculature(perivascular fibroblasts, coronary smooth muscle cells and endothelialcells) (von Gise & Pu, 2012).

Disruption of numerous mediators of the EMT process have beendemonstrated to cause congenital heart defects. For example, theepicardial transcription factor Wt1 is enriched in the embryonic heart,and is required for embryonic coronary artery formation and essentialfor epicardial EMT (Moore, McInnes, Kreidberg, Hastie, & Schedl,1999). Wt1 contains a DNA binding domain with a proline-glutaminerich region capable of regulating transcription. Correspondingly,two of the major mediators of EMT, Snail (Snail) and E-cadherin(Cdh1), are controlled through direct transcriptional regulation byWt1, thereby affecting cell differentiation (Martinez-Estrada et al.,2010). Furthermore, Guadix et al. (2011) demonstrated Wt1binding regions in the Raldh2 promoter and the direct transcriptionalcontrol of Raldh2 expression by Wt1 in epicardial cells. Thedevelopmental deficiencies of mice with a homozygous deletionfor Wt1 were reported by Kreidberg et al. (1993). Even epicardialspecific Wt1 mutants die from cardiovascular defects betweenE16.5 and E18.5 (Martinez-Estrada et al., 2010). Similarly, Tbx18deficient mouse embryos exhibit structural and functional defectsof the epicardium and coronary vessels (Wu, Dong, Regan, Su, &Majesky, 2013).

Many of the signaling pathways associated with EMT have beendemonstrated to be required for proper embryonic heart development.

Both TGF-β1 and TGF-β2 deficient mice develop severe cardiac defects(Bartram et al., 2001; Letterio et al., 1994). Similarly, BMP2 deficiencyis embryonic lethal, and BMP2-deficient embryos exhibit malformedcardiac cushions and abnormal cardiac development in the exocoelomiccavity (Ma, Lu, Schwartz, & Martin, 2005; Zhang & Bradley, 1996).Interrupted Notch signaling disturbs smooth muscle differentiationof EPDCs in coronary vascular development, resulting in EPDCswhich surround developing arteries but do not provide sufficientsupport for normal vascular development (Grieskamp, Rudat, Ludtke,Norden, & Kispert, 2011). Likewise, deficiency of either FGF or PDGFattenuates EMT, EPDC invasion into the myocardium, and vesselcell differentiation during coronary vascular formation (Pennisi &Mikawa, 2009; Smith et al., 2011). Using a conditional β-catenin-deletion mutant, Zamora, Manner, and Ruiz-Lozano (2007) demon-strated similar impairment in coronary artery formation due to failedepicardial expansion, blunted epicardial invasion into the myocardium,and impaired differentiation into smoothmuscle cells. Jing et al. (2016)demonstrated the importance of HIF-1α mediated Snail expression inepicardial Tbx18+ cells for smooth muscle cell differentiation; blockingnuclear accumulation of HIF-1α with 2-methoxyestradiol attenuatedhypoxia induced Snail and smooth muscle marker expression in thesecells. Similarly, proper RA signaling is required for epicardial EMT forcoronary arteriogenesis, as demonstrated by reduced β-catenin andFGF2 expression and abnormal arterial branching in tissue specificretinoic acid receptor mutant (Merki et al., 2005). In this study,arteriogenesis was found to be impaired due to a deficiency inretinoid-dependent Wnt signaling. Furthermore, conditional epicardialdeletion of retinoid X receptors (RXR) in development using theGata5-Cre caused thinning of the subepicardial compartment resultingin a hypoplastic myocardium. The activation of epicardial cells duringdevelopment is also suggested to cause trophic factor productionwhich contributes to the proliferation of cardiomyocytes. Conditionedmedia from epicardial cell cultures causes proliferation of primaryfetal cardiomyocytes. Interestingly, trophic factor production isaugmented by RA treatment, and inhibited by a retinoid receptorantagonist (Chen et al., 2002). Reduced trophic factor productionmay have also been responsible for the hypoplastic myocardiumobserved by Merki et al. (2005) in their retinoic acid receptor (RAR)mutant mice.

Outside of growth factors and cytokines, miRNAs and primary ciliawhich regulate EMT also play a role in cardiac development. To thisend, Singh, Lu, Massera, and Epstein (2011) demonstrated thatregulation ofmiRNAs byDicer is critical for epicardial EMT and coronarysmooth muscle cell differentiation during development. In this study,epicardial-specific deletion of Dicer impaired EMT, epicardial cellproliferation and coronary vessel development, resulting in 100%postnatal fatality. Likewise, multiple human ciliopathies have cardiacabnormalities such as dextrocardia, aortic stenosis, and septum defects(Koefoed et al., 2014). Mutations in cilia proteins in animal modelsresults in congenital heart defects (Clement et al., 2009; Li et al.,2015). Specifically, Ift88-null mice develop hearts with ventriculardilation, reducedmyocardial trabeculation, and outflow tract abnormal-ities (Clement et al., 2009). In this study, it was demonstrated that Hhsignaling is associated with the cilium in cardiomyocytes, suggestingits loss could be responsible for the observed cardiac phenotype ofthese Ift88-null mice. Furthermore, both endocardial and epicardialcells in the heart are ciliated. However, under the influence ofhigh shear stress (as in cardiac development), cells become devoid ofcilia as the pressure signals the microtubules in these organelles todissociate (Van der Heiden et al., 2006). Interestingly, FGF receptorknockdown or FGF inhibition downregulates expression of coreciliary proteins and reduces the ciliary length during development(Neugebauer, Amack, Peterson, Bisgrove, & Yost, 2009). Importantly,the absence of ciliary function in the embryonic mouse heart coincideswith endocardial–to–mesenchymal transition (EndMT) (Egorovaet al., 2011).


7. Cardiac repair

7.1. Insight from regenerative models

Until recently, the dogma dictating an incapacity for completemammalian cardiac healing was in sharp contrast with a completeregeneration in non-mammalian vertebrate models, such as thatdemonstrated by zebrafish from cardiac apex resection (Jopling et al.,2010; Poss, Wilson, & Keating, 2002). Notably, two surgical models ofmammalian cardiac regeneration in the neonatal mouse have beendeveloped (Blom, Lu, Arnold, & Feng, 2016; Haubner et al., 2012;Porrello et al., 2011). However, the regenerative capacity in neonatalmicewas retained only in the early neonatal period, andwas lost shortlyafter birth by P7. Evidence fromcoronary corrosion casts indicates that avascular response restores perfusion during neonatal heart regenera-tion (Porrello et al., 2013). Furthermore, EMT genes are upregulatedpost-cardiac apex resection in the neonate, implicating epicardial EMTin neovascularization (Porrello et al., 2011). Indeed, data from zebrafishmodels designates the epicardium as the cellular source of the regener-ated vascular compartment during cardiac regeneration, corroboratingthis hypothesis (Kikuchi et al., 2011); in this study, Cre-mediatedlineage tracing of Tcf21+ (epicardin) epicardial cells demonstrateddifferentiation into mesenchymal perivascular cells, but notcardiomyocytes. In other experiments, the zebrafish epicardium re-expressed Wt1, Tbx18 and aldh1a2 after cardiac injury, and Wt1+

cells were shown to differentiate into perivascular cells (Gonzalez-Rosa, Peralta, & Mercader, 2012; Lepilina et al., 2006). Disruption ofmediators of EMT including PDGF and FGF disturb neovascularizationduring zebrafish heart regeneration, demonstrating the importance ofEMT mediated repair (Kim et al., 2010; Lepilina et al., 2006). Similarly,treatment with the Hh antagonist cyclopamine blunts the epicardialregenerative capacity post-apex resection, and transplantation of Shh-soaked beads to the ventricular base stimulates epicardial regeneration(Wang, Cao, Dickson, & Poss, 2015). Furthermore, hyaluronic acid (HA)is a glycosaminoglycan known for its role in formation of granulationtissue for wound healing. Recently, HA and its receptor Hmmr havebeen implicated in epicardial EMT for cardiac remodeling. Knockdownof Hmmr in a zebrafish ventricular apex resection model reducedepicardial cell migration and coronary vascular formation, causingpermanent fibrosis (Missinato, Tobita, Romano, Carroll, & Tsang, 2015).

7.2. EPDCs and cardiac repair

In adult mammals, this dormant fetal growth program is reactivatedafter MI by stimuli such as increased wall stress, which activatesmechanical stretch receptors, triggering immediate early genes suchas c-jun, c-fos, and c-myc (Gidh-Jain, Huang, Jain, Gick, & El-Sherif,1998; Reiss et al., 1993; Sadoshima, Jahn, Takahashi, Kulik, & Izumo,1992). Hypoxia from injury induces hypoxia-inducible factor-1 alpha(HIF-1α), which is responsible for the upregulation of VEGF and otherpro-angiogenic factors that are implicated in epicardial EMT (Forsytheet al., 1996; Tao, Doughman, Yang, Ramirez-Bergeron, & Watanabe,2013). Indeed, fetal epicardial markers are also re-expressed after MIin adult mice. This is particularly important in subsets of EPDCs thatexpress Wt1 and Tbx18, markers of embryonic cardiovascular progeni-tors (vanWijk, Gunst, Moorman, & van den Hoff, 2012). These cells arethought to contribute to angiogenesis and stem cell recruitment post-MI through paracrine effects by up-regulating molecules such as FGF,PDGF, and monocyte chemoattractant protein 1 (MCP-1) (Zhou et al.,2011). Within the infarcted myocardium, some endothelial cells alsoexpress Wt1. Since Wt1 promotes endothelial cell proliferation, theWt1+ endothelial cells may contribute to the angiogenic response(Duim, Kurakula, Goumans, & Kruithof, 2015). Recently, post-MIEPDCs were also shown to express the ectoenzyme CD73 on their cellsurface, which can convert extracellular ATP and NAD to adenosine(Hesse et al., 2017). Excess extracellular adenosine stimulated the

release of cytokines like IL-6, IL-11, and VEGF. These authors alsodemonstrated the release of the fetal protein tenascin-C from EPDCs,which stimulated EPDC migration. While this milieu may supportangiogenesis, it may also contribute by polarizing the inflammatoryresponse to support cardiac repair.

The immune response has been demonstrated to be important inboth the acute and long-term responses to MI (Frangogiannis, 2014).In general, the innate system is activated in the acute setting and playsa role in wound clearance and scar formation. Overactive adaptiveimmune responses however, are thought to accentuate deleteriousremodeling and contribute to heart failure. For example, the importanceof macrophages for cardiac repair was demonstrated by failure of theneonatal cardiac regenerative capacity inmice depleted ofmacrophagesby administration of clodronate liposomes pre and post-MI (Auroraet al., 2014). In a recent study, the epicardium was shown to modulatethe adaptive immune response via epicardial Hippo signaling (Ramjeeet al., 2017). Indeed, YAP/TAZ signaling, which activates Wnt and IGFpathways, was shown to be required for neonatal cardiac regeneration(Xin et al., 2013). Cardiac specific Yap knockout mice retained anextensive fibrotic scar 26 days after infarction induced on postnatalday 2. Ramjee et al. (2017) further demonstrated the critical importanceof epicardial YAP/TAZ in suppressing post-infarct inflammatoryresponses by recruiting T-regulatory cells. In this study, loss ofepicardial Hippo signaling by tamoxifen mediated Yap and Taz deletion(Wt1CreERT2/+Yapf/fTazf/f) did not affect expansion of EPDCs, but didresult in reduced interferon expression and a profound inflammatoryresponse leading to increased fibrosis and mortality post-MI.

Beyond paracrine signaling, Wt1+ epicardial cells are able torecapitulate their embryonic profile and undergo EMT. This embryonicreprogramming is responsible for detachment, inward movement andmitosis of transitioning epicardial cells post-MI. Once transitioned,differentiated EPDCs lose Wt1 expression (Moore et al., 1999).Post-MI, epicardial cells express mesenchymal markers rather thanepicardial ones, implying their mesenchymal transition (Zhou et al.,2011). The ability of epicardial cells to undergo EMT in humanswas confirmed in isolated primary adult epicardial cells grown inculture, which demonstrated the potential to undergo EMT andre-differentiate (van Tuyn et al., 2007). Most authors agree that EPDCspossess the capacity to re-differentiate into vascular smooth musclecells and myofibroblasts, but less likely endothelial cells. Interestingly,however, some authors have suggested that transitioning epicardialcells may re-differentiate into cardiomyocytes. For example, van Wijket al. (2012) usedWt1Cre;R26R mice to show that EPDCs form a limitednumber of cardiomyocytes 1 and 3 months post-MI. However, thesecardiomyocytes did not display the typical cardiomyocyte architecture,but rather co-expressed the cardiomyocyte markers, cardiac troponinI and SERCA2a. Whether these cells indeed differentiate intocardiomyocytes during cardiac development or post-MI is the subjectof debate (Cai et al., 2008; Manner, 1999; Smart et al., 2011; Zhouet al., 2012). Regardless of the differentiation potential of EPDCs intocardiomyocytes, the endogenous capacity of adult EPDCs is clearly notsufficient to replace the lost cardiomyocytes post-MI.

7.3. EPDC and cardiac fibrosis

As reviewed by Fang, Xiang, Braitsch, and Yutzey (2016), numeroussources have also demonstrated the fibrotic differentiation potential ofEPDCs in both development and disease. It is possible that differentmediators of EMT support the differential linage determination ofEPDCs. For example, as mentioned previously, PDGFRβ activationsupports coronary vascular smooth muscle cell differentiation, whilePDGFα activity encourages fibroblast fate specification (Smith et al.,2011). Interestingly, EPDC derived, and not bone marrow cell derived,cardiac fibroblasts are thought to contribute the majority of cardiacfibroblasts to the post-ischemic myocardium (Ruiz-Villalba et al.,2015). To discern this, irradiatedWt1Cre-YFP+ mice were transplanted


with mRFP bone marrow and subject to MI. Although both YFP+ andRFP+ cells infiltrated the myocardium in response to MI, YFP+ EPDCsrepresented the majority of retained fibroblasts post-MI. However,these fibroblasts were suggested to be derived mainly from embryonicEPDC precursors that differentiated during development which subse-quently proliferate post-MI in response to signals such as SDF-1, ratherthan by adult epicardial EMT. Regardless of the time of differentiation,this fibrotic response post-MI is critical to preserving cardiac function.In a recent study, the importance of EPDCs in fibrosis post-MI wasdemonstrated. In this study, disruption of Wnt/β-catenin signaling inepicardial cells usingWt1-Cre-mediated excision of β-catenin impairedepicardial EMTmediated fibroblast differentiation, andworsened cardi-ac dysfunction post-MI (Duan et al., 2012). As mentioned previously,the initial fibrotic response is important for scar formation to mitigatefurther damage and prevent ventricular rupture. However, long-termfibrotic responses are associated with progression to heart failure.Since the activation of epicardial cells post-MI is transient (van Wijket al., 2012), perhaps the epicardial fibrotic response is beneficial forthe acute phase of healing. Indeed, as exemplified by Zhou et al.(2011), most studies indicate that epicardial activation reduces infarctsize and LV stiffness post-MI.

8. EMT for post-MI repair

8.1. Gain of function approaches

Enhancing the EMT process may augment EPDC signaling, as well asinfarct and peri-infarct angiogenesis in the adult, thereby providing atarget for therapeutic development (Fig. 3). In pre-clinical animal

Fig. 3. Schematic diagram underlying epicardial EMT post-MI. Epicardial cells are activated by pfurther support epicardial activity. These paracrine factors also support stem cell recruitment wthe immune response. Post-MI, EPDCs re-differentiate into smooth muscle cells, myofibroblasadipocytes. This process can be enhanced by administration of exogenous materials which mscaffolds, and hydrogels. Exogenous material may also be delivered through ultrasound media

models, overexpression approaches for growth factors and cytokineshave demonstrated the potential of enhancing EMT for cardiac repair.Our laboratory recently demonstrated that the contribution of EPDCprogenitors can be augmented in the adult by other factors related toembryogenesis, including stem cell factor (SCF) (Xiang, Liu, Lu, Jones,& Feng, 2014). Overexpression of membrane-associated human SCF inthe murine myocardium augmented cardiac repair. The breakdown ofthe epicardial basement membrane post-MI allowed SCF expressed bythe myocyte to interact with the c-Kit receptor on the epicardium.This induced the expression of the key EMT regulator, TGF-β, enhancingepicardial activation and the production of EPDCs, contributing toarteriogenesis (Xiang et al., 2014). Furthermore, using a lineage tracingapproach to fate map Wt1+ cells (ROSAmTmG;Wt1CreER mice), wedemonstrated the transdifferentiation of EPDCs into SMA+ cells(myofibroblasts or vascular smooth muscle cells), but notcardiomyocytes or endothelial cells. Importantly, cardiac specific SCFoverexpression decreases infarct size, inhibits cardiac remodeling, andimproves myocardial function and animal survival post-MI (Xianget al., 2009). Interestingly, Zakharova, Nural-Guvener, and Gaballa(2012) found thatmost c-Kit+ cells derived from atrial explants expressbothWt1 andNotch in culture, and these cells undergo EMTuponNotchstimulation, supporting the role of Notchmediated epicardial activationin cardiac repair. Not surprisingly, using a transgenic Notch reportermouse, others demonstrated that MI amplifies Notch-activated EPDCs,which support a reparative response (Russell et al., 2011). Interestingly,a novel population of c-Kit+ cells termed cardiac telocytes have beendiscovered to be present in large numbers in the epicardial compart-ment (Popescu et al., 2010). These cells are also positive for PDGFRβand the progenitor cell marker CD34. Cardiac telocytes are thought to

ost-MI stimuli to undergo EMT and become EPDCs. EPDCs release paracrine factors whichhile enhancing angiogenesis, arteriogenesis, cardiomyocyte proliferation, and modulatingts, pericytes, and potentially contribute endothelial cells, fibroblasts, cardiomyocytes, anday contain growth factors and cytokines using epicardial patches, extracellular matrix

ted microbubble delivery. All of these processes support cardiac repair post-MI.


contribute to intercellular communications via atypical cellularjunctions and shedding of numerous microvesicles and multivesicularbodies which may contain RNAs and proteins (Gherghiceanu &Popescu, 2012). Work by Zhao and colleagues has revealed that thenetwork of cardiac telocytes in the infarct area are destroyed post-MI,but transplantation of telocytes significantly improves vessel densityin the infarct and peri-infarct zone, reduces infarct size, and improvesmyocardial function (Zhao et al., 2013).

8.2. Roles of growth factors and cytokines

Hypoxia causes epicardial and myocardial secretion of many of thefactors which support EMT post-MI. While these factors diffuse acrossthe ventricular wall and affect adjacent myocytes, some factors arereleased into the pericardial fluid. Evidence suggests that trophic factorswithin pericardial fluid from MI patients supports epicardial cellproliferation andWt1 expressionwhen injected into the pericardial cav-ity of non-infarcted mouse hearts (Limana et al., 2010). Subsequently,the same group identified clusterin, a secreted glycoprotein, in the peri-cardial fluid from patients with acute MI (Foglio et al., 2015). Clusterintreatment alone after acute MI in vivo supported epicardial EMT whichenhanced arteriogenesis and reduced cardiomyocyte apoptosis. Similar-ly, exogenous administration of several other factors which support epi-cardial EMT have been demonstrated to promote repair. For example,intramyocardial injection of HGF complexed to an IgG carrier enhancedvascular integrity post-MI in a rat model (Rao et al., 2015). Similarly,intramyocardial injection of a constitutively active plasmid hybridencodingHIF-1α andherpes simplex virus VP16 enhanced angiogenesisand improved regional blood flow (Shyu et al., 2002). Although HIF-1αadministrationwas determined to enhance VEGF expression, this groupdid not discern the cellular source of improved angiogenesis; whethernew vessels came from an epicardial source or pre-existing vesselswas not investigated. The role of VEGF in direct epicardial activationpost-MI has been confirmed using synthetic modified RNA (modRNA).In this study, intramyocardial injection of VEGF-A modRNA resulted inepicardial progenitor cell mobilization into the myocardium (Zangiet al., 2013). Cultured EPDCs treatedwith VEGF-AmodRNAdemonstrat-ed endothelial expression characteristics, and treated hearts had im-proved Wt1+ cell proliferation and reduced scar area. Lineage tracingwith Wt1CreERT2 mice demonstrated EPDC expression of both cardio-myocyte and endothelial markers after VEGF-A modRNA treatment.

Contrastingly, while most of these factors have been demonstratedto improve cardiac repair, a recent study elucidated a deleteriousrole of growth factor mediated epicardial activation (Zangi et al.,2017). While many EPDCs contribute to intramyocardial vessels, someEPDCs differentiate into fat cells. Using Wt1-CreER;Rosa26RFP/+ micefor lineage tracing, Liu, Huang, et al. (2014) demonstrated co-immunostaining of epicardial cells with perilipin, an adipocyte marker.Furthermore, in the study by Zangi et al., administration of IGF-1 afterMI supported the differentiation of EPDCs into epicardial adipocytes.Although the effect of this differentiation process on infarct size andfibrosis was not discussed, the authors suggested that this type ofEPDC differentiation may contribute to worsening coronary arterydisease (Zangi et al., 2017). While others have demonstrated the abilityof IGF-1 to support cardiac repair, preserving both ejection fraction andcapillary density (Koudstaal et al., 2014), this recent data should beconsidered when evaluating therapies using IGF-1.

A body of research surrounding Tβ4 has also demonstrated itspotential as a biological stimulus for adult epicardial activation. Cardiacspecific, Nkx2.5Cre-driven shRNA knockdown of Tβ4 in mice resulted inembryonic lethality at E14.5–15.5 with abnormal heart and coronaryartery development, suggesting an essential role of Tβ4 in coronaryformation during embryogenesis (Smart et al., 2007). Tβ4 was alsoshown to stimulate the outgrowth of adult epicardial cells in explantculture, which is typically lost after the neonatal period. Subsequently,the same group demonstrated that pretreatment of Tβ4 in adult mice

by intraperitoneal injection augments neovascularization and stabilizesvascular plexus formation post-MI (Smart et al., 2011). In this study,EPDCs were found to migrate to peri-vascular areas and expressPECAM and α-smooth muscle actin. Furthermore, pretreatment of Tβ4has been shown to increase the timewindowof neonatal heart regener-ation to postnatal day 7, by supporting Wt1+ EPDC migration into themyocardial region (Rui et al., 2014). Unfortunately, however the reportby Zhou et al. (2012) questioned the ability of Tβ4 to promote EPDCmobilization; the report suggested that Tβ4 supports EPDC activationand differentiation into smooth muscle cells, but these cells do notmigrate substantially into the myocardium. These authors alsostipulated that Wt1+ EPDCs activated by Tβ4 treatment post-MI donot differentiate into cardiomyocytes or endothelial cells, but mayprovide beneficial paracrine factors for repair (Zhou et al., 2012). Thisdiscrepancy may, in part, be explained by the epigenetic regulationand chromatin accessibility of key epicardial, EMT and cardiomyocytegenes, which were found to be induced by Tβ4 pre-treatment.

Recent evidence implicates Tβ4 in epigenetic regulation and chroma-tin accessibility. Brahma-related gene 1 (BRG1) is an ATPase subunit es-sential for SWI/SNF mediated chromatin-remodeling. Using chromatinimmunoprecipitation-sequencing, Vieira et al. (2017) demonstratedBRG1-SWI/SNF activity at evolutionary conserved regions of the Wt1locus, causing chromatin remodeling. Not only did this study show thatBRG1 is required for Wt1 activation in EPDCs, authors also providedevidence for Tβ4 as a co-activator to enhance Wt1 transcription. Tβ4co-immunoprecipitated with BRG1, and its expression pattern wasspatiotemporally equivalent to BRG1. Priming of Tβ4 knockout micewith Tβ4 augmented Wt1 reactivation post-MI, confirming the require-ment for Tβ4 in epicardial response to stress (Vieira et al., 2017).

However, in sharp contrast to the study by Smart et al. (2007), nei-ther global nor cardiac specific Tβ4 deficient mice show any abnormal-ities in embryonic heart development, coronary artery development oradult cardiac function (Banerjee et al., 2012). These authors suggest thatthe difference between these two studies is most likely due to the off-target effects of the shRNA approach used by Smart et al. (2007).Additionally, Tβ4 treatment in pigs before ischemia or after reperfusiondoes not appear to be cardioprotective in the short term i.e., within 30 hof ischemia/reperfusion injury (Stark et al., 2016). Furthermore, a phaseII clinical trial on the safety and efficacy of Tβ4 treatment in patientswith acute MI was suspended in 2015, citing that the drug productmanufacturer was not GMP compliant (NCT01311518). Thus, thepotential of Tβ4 as a treatment of MI remains to be determined.

Epicardial expression of trophic factors appears to be critical not onlyfor mediating angiogenesis, but also cardiomyocyte survival anddivision to support cardiac repair. In 2011, the importance of EPDCs asa contributor to fibroblasts and smooth muscle cells, and paracrinemediators for adult cardiac repair was demonstrated (Zhou et al.,2011). MI induced EPDC mesenchymal change and EPDC conditionedmedium stimulated endothelial cell proliferation and vessel assembly.Epicardial cells secreted proangiogenic factors such as VEGFa, FGF,PDGF and MCP1. Subsequently, epicardial cells were found to producefollistatin-like 1 (Fstl1), a cardiogenic mitogen (Wei et al., 2015). Inthis study, collagen patches treated with conditioned media fromepicardial cells significantly improved cardiac function as compared tocontrol patches. Similarly, collagen patches conditioned with Fstl1significantly improved cardiac function, but this effectwas not observedwith Fstl1-transgenicmice that overexpressed Fstl1 in themyocardium.These results indicate that epicardial specific Fstl1 supports myocyteproliferation and cardiac repair. Improved function was also associatedwith increased vascularization (Wei et al., 2015).

8.3. Other mediators of epicardial activation

Beyond growth factors and cytokines, the role of non-coding RNA inEMT has prompted investigation into modulating these factors to sup-port cardiac repair. As reviewed elsewhere, manymiRNAs and lncRNAs


have been investigated for their role in supporting angiogenesis andcardiomyocyte survival and proliferation (Guo, Luo, Liu, & Xu, 2017).More recently, however, a role formiRNAs in specifically regulating epi-cardial EMT to improve post-MI recovery has been demonstrated(Seeger et al., 2016). The let-7 family of miRNAs is composed of 13members and regulates numerous functions including cell proliferationand differentiation (Roush & Slack, 2008). The expression of let-7 familymembers was increased post-MI, and this increase could be blocked byadministration of a let-7 anti-miR. Anti-miR administration supportedepicardial cell recruitment, EMT, and cardiac functional improvement(Seeger et al., 2016). Contrastingly, in another investigation, miR-21supported Timp-1 expression and E-cadherin loss in TGF-β inducedEMT of EPDCs while anti-miR for miR-21 blocked epicardial EMT(Bronnum et al., 2013). As the understanding of ncRNA is still in itsinfancy, it is expected that modulation of other ncRNA targets willdemonstrate efficacy in supporting epicardial EMT for cardiac repair inthe future.

Like the emerging investigations in ncRNA and cardiovasculardisease, the role of the primary cilium in cardiac EMT has not beenwell investigated post-MI. As mentioned above, the primary ciliumhas recently been established as a mediator of both EMT and cardiacdevelopment. In a recent investigation from our laboratory, weuncovered a role for the primary cilium in cardiac repair (Blom, Lu,Kim, & Feng, 2016). Using a model of MI in postnatal day 7 mice, wedemonstrated the efficacy of promoting ciliary disassembly inprolonging the age-dependent limitation on cardiac regeneration. Inthis study, shRNA for Ift88, a protein vital for ciliary function, supportedepicardial EMT and cardiac regeneration in mice beyond the neonatalregenerative window. Our recent work also confirms the importanceof the primary cilium in mediating epicardial EMT in the adult micepost-MI, and suggests the primary cilium as a target for supportingpost-MI repair (Blom et al., unpublished observations).

8.4. Cells and tissue engineering

As discussed above, transplantation of progenitor cells to themyocardium has shown benefit in pre-clinical models, but has metchallenges in clinical application. In an early study by Winter et al.(2007), transplantation of human adult EPDCs into infarcted mousehearts preserved ventricular function at 2 weeks, and up to 6 weeks-post MI. Engrafted EPDCs expressed α-smooth muscle actin and vonWillebrand factor, but not markers of cardiomyocytes. Also, engraftedEPDCs did not necessarily integrate into vessel walls, and few weredetected at the long-term time point of 6 weeks; thus, authorssuggested that the contribution of the EPDCs to vasculogenesis wasmostly mediated via a paracrine mechanism. In a subsequent study,co-transplantation of human EPDCs and cardiomyocyte progenitorcells further improved cardiac function beyond the transplantation ofone cell type alone (Winter et al., 2009). These authors speculatedthat this effect was the result of communication between co-transplanted cells causing complementary secretion of paracrinefactors.

Interestingly, others have demonstrated that the transplantation ofprogenitor cells to the ischemic myocardium provides superiorreparative effects when placed epicardially as compared to injectionintramyocardially. The epicardial application of a cardiogenic scaffoldharboring human cardiac-derived cardiomyocyte progenitor cells in aporous 3D printed hyaluronic acid/gelatin based biomaterial supportscell engraftment and vascular differentiation, as well as long-termsurvival in a mouse model of MI (Gaetani et al., 2015). Engrafted cellsexpressed troponin I and CD31 at 1 month post-engraftment. Tanoet al. (2014) also demonstrated that epicardial placement of mesenchy-mal stromal cell-sheets produces enhanced production of the paracrinefactors IGF-1,MMP-2 andHIF-1α, molecules expressed during epicardialEMT. Mesenchymal sheet cells expressed PECAM1 on day 3 post-placement, however these cells did not appear to migrate into

the heart. Furthermore, these cells did not differentiate into cardiaccells expressing troponin T, implicating the paracrine mediators in theobserved improvement in cardiac function. This type of cell transplanta-tion strategy has been employed in a phase I clinical trial to treat cardio-myopathy patients using autologous cells. Although not all patients inthis trial experiencedMI as their primary injury, the cell-sheet implantsappear to possess a promising safety profile and support some function-al recovery (Miyagawa et al., 2017). Larger studies are warranted tofurther explore both the use of cell-sheet therapy for cardiac repair inthe clinical setting, particularly post-acute MI, and the mechanism bywhich cell-sheets produce their effects.

Although the implementation of cells and genetic material has beencontroversial in large animal models and in the clinical setting, recentdevelopment in biomaterials such as patches and hydrogels offerspromise for continued development of the field. For example, whileTβ4 has demonstrated conflicting long-term results in larger animalsand through one time administration via IP injection or intramyocardialdelivery, the use of biomaterials seems to remarkably improve itseffectiveness. Using ultrasound-targeted microbubble delivery ofhuman Tβ4 under a transposon plasmid system, Chen, Shimoda, Chen,and Grayburn (2013) demonstrated significant Tβ4 overexpression inrat hearts for three months. Using this delivery method, Wt1+ progen-itor cells increased in number, and began to express cardiac troponin Tin themyocardium.Microbubble Tβ4 delivery also supportedprolongedangiogenesis and arteriogenesis, measured by alpha-smooth muscleactin, VEGF, Tie-2 and PECAM mRNA, as well as coronary artery andcapillary density. Finally, Tβ4 induced proliferation of cardiomyocytesin the long-term (3 months), indicating self-renewing potential.Similarly, Chiu, Reis,Momen, and Radisic (2012) enhanced Tβ4 deliveryusing a biomaterial approach by controlled release from collagen-chitosan hydrogels. Hydrogel type materials had been previouslydemonstrated to impart structural reinforcement, but were unable toprevent deleterious remodeling post-MI (Rane et al., 2011). Controlledrelease of Tβ4 with a collagen-chitosan hydrogel improved vasculariza-tion and reduced tissue loss (Chiu et al., 2012). Similarly, Boopathy et al.(2015) used hydrogels to administer the Notch ligands, which resultedin improved cardiac function and angiogenesis post-MI. Hydrogelmediated-Notch ligand administration significantly augmented thenumber of Ki67-positive, lectin-positive endothelial cells. Anothergroup demonstrated that an epicardially placed hydrogel containingbFGF reduced infarct size and increased microvascularity in a rabbitmodel of MI (Fujita et al., 2005).

Furthermore, epicardial based biomaterial patches have gainedrecognition for their supportive capabilities post-MI. In addition to thestudy on Fstl1 mentioned above (Wei et al., 2015), others have demon-strated the utility of epicardial patching to aid in the delivery ofsubstances to support epicardial mediated repair. Beyond the directmechanical benefit of patching the epicardium, which provides struc-tural support and restricts ventricular wall dilation (Serpooshan et al.,2013), patches containing cells or cytokines and growth factors areable to further enhance epicardial mediated repair. For example,bFGF-enriched extracellular matrix (ECM) biomaterial patches in a ratMI model improved long term ejection fraction beyond the ECM patchalone (Mewhort, Turnbull, Meijndert, Ngu, & Fedak, 2014).

A recent study in rats demonstrated the ability of a mesenchymalstem cell-loaded cardiac patch to secrete HIF-1α, Tβ4, VEGF and SDF-1to support activation of Wt1+ EPDCs, which migrated deep intothe myocardium and differentiated into smooth muscle cells, andpotentially a small number of cardiomyocytes (Wang et al., 2017).Unfortunately, as has been argued previously, the sparse availability ofautologous cells for transplantation may limit the feasibility of thistype of therapeutic approach. Other cell types for cardiac patches canbe primed for directed differentiation such that they support repair.For example, Zhao et al. (2017) demonstrated that stimulation ofthe Wnt and RA pathways can direct cardiac progenitor cells to an epi-cardial cell fate, and these cells are supportive of smooth muscle cell


differentiation rather than a fibrotic response. In this study, humanpluripotent stem cells (hPSCs) were directed to differentiate intoTbx18+/Wt1+ cobblestone shaped epicardial like cells by RA and Wntrespectively; these cells were then induced to transform to amesenchy-mal phenotype using bFGF and TGF-β1 administration.While the use ofprimed cells in cardiac patches is gaining popularity, the ability to directhPSCs into desired cell types is becoming increasingly feasible. Recently,Witty et al. (2014) and Iyer et al. (2015) were also able to differentiatehPSCs into epicardial-like cells via stimulation with BMP, Wnt and RApathways. Combining this type of approach with hydrogels or patchescould provide a sufficient source of accessible cells whichwould possessthe characteristics of EPDCs to support cardiac repair.

8.5. Small molecules

It has become increasingly apparent that small molecularcompounds may be efficacious in aiding with treatment modalities forvarious types of disease. Recent studies have explored the use of smallmolecules to enhance epicardial differentiation and EMT in rodentmodels of MI. Sasaki, Hwang, Nguyen, Kloner, and Kahn (2013) showedthat a small molecule Wnt signaling modulator ICG-001 decreasesβ-catenin/CREB binding protein (CBP) interaction, while concomitantlyincreasing the β-catenin/p300 interaction. They further demonstratedthat ICG-001 promotes epicardial EMT and improves cardiac functionin rats with MI. In the study by Zhao et al. (2017) the Wnt pathwaywas stimulated with the use of a small molecule GSK-3 inhibitor,CHIR99021, to induce Wnt signaling and epicardial differentiationfrom hESCs. Similarly, Bao et al. (2016) recently demonstrated 95%Wt1+ epicardial cell differentiation from hPSCs using three GSK-3small molecule inhibitors: CHIR99021, CHIR98014 and BIO-acetoxime.This group additionally demonstrated efficacy of using TGF-β receptorinhibitors A83-01, RepSox, SB505124, and SB431542 in expanding theepicardial cell population. Importantly, these epicardial cells can beseeded on cardiac-fibroblast-derived extracellular matrix patches. In amouse-MI model, application of these epicardial seeded patches ontothe heart surface resulted in significant EMT and smooth-muscle-likeand fibroblast-like cell differentiation within the mid-myocardium(Bao et al., 2016). However, longer-term efficacy of this patch onmyocardial function post-MI remains to be determined. Additionally,primary human EPDCs have been used to screen 7400 structurallydiverse compounds that are known to modulate a broad range ofbiological targets (Paunovic et al., 2017). The phenotypic screens haveidentified small molecules that promote human EPDC proliferation. Itis expected that some of these small molecules can be further modifiedto optimize their metabolic and pharmacokinetic properties for in vivostudies.

9. Conclusions

Over the last two decades, the understanding of the contribution ofthe epicardium and its role in cardiac development and repair hasincreased dramatically. Seminal work using zebrafish demonstratedthe importance of EPDCs in the regenerating myocardium. Morerecently, molecular tools and fate-mapping have demonstrated thecontribution of EPDCs to the developing mammalian heart. This led tothe discovery of EPDC activation in adult mammals upon cardiac injury.Efficacies of augmenting epicardial EMTpost-MI have been demonstrat-ed using growth factors and cytokine mediators, as well as innovativebiomaterials in animal models (Fig. 3). Similarly, the novel understand-ing of different levels of gene regulation has revealed the involvement ofncRNA in this process. miRNAs and lncRNAs play a pivotal role incardiogenesis and understanding their roles in adult epicardial EMTmay provide additional targets for therapeutic intervention. Likewise,the primary cilium, which regulates signaling by EMT mediators, mayserve as a promising target to augment epicardial responses post-MI. Al-though these promising advances have been made in the search for

therapeutics to support cardiac repair, a suitable treatment has yet tobe successful in large clinical trials. Continued investigation into thepower of the epicardium to both mediate angiogenesis and supporttrophic factors for cardiac repair and/or regeneration may provide thekey to unveiling a cure for heart failure.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

Work in the authors' laboratory is supported by theHeart and StrokeFoundation of Canada (G-17-0018659), the Canadian Institutes ofHealth Research (MOP-142383), and the Natural Sciences and Engi-neering Research Council of Canada (RGPIN-2017-06581). Thesefunding agencies had no role in decision to publish or preparation ofthe manuscript.

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